www.arch-anim-breed.net/59/417/2016/ doi:10.5194/aab-59-417-2016
© Author(s) 2016. CC Attribution 3.0 License.
Open Access Archives Animal Breeding
Excessive amount of lactose in the diet of two-week-old
calves induces urinary protein changes
Alicja Dratwa-Chałupnik, Małgorzata O˙zgo, Adam Lepczy ´nski, Agnieszka Herosimczyk, and Katarzyna Michałek
Department of Physiology, Cytobiology and Proteomics, Faculty of Biotechnology and Animal Husbandry, The West Pomeranian University of Technology, Szczecin, Poland
Correspondence to:Alicja Dratwa-Chałupnik ([email protected])
Received: 10 May 2016 – Revised: 12 September 2016 – Accepted: 19 September 2016 – Published: 13 October 2016
Abstract. The present paper was undertaken to analyse and identify urinary proteins that were significantly altered in urine of calves in response to short-term administration of milk replacer with lactose addition. We used 2-D electrophoresis combined with matrix-assisted laser desorption/ionisation and time-of-flight (MALDI-TOF) mass spectrometry. Of all spots analysed, four showed significantly decreased abundance: alpha-1-antiproteinase (A1AT), serotransferrin (TF), sex hormone-binding globulin (SHBG) and cytochrome P450 2E1 (CYP2E1). One displayed an increased abundance: adenosine triphosphate (ATP)-citrate synthase. The changes in abundance of SHBG and CYP2E1 proteins were caused by the direct effect of an oversupply of sugar, while A1AT, TF and ATP-citrate synthase showed altered abundance probably due to indirect effects. The results of this study confirmed that calves’ urine is a very precious biological material to evaluate the renal function, and it may be valuable in veterinary and zootechnical diagnostics.
1 Introduction
Understanding biochemical processes that take place within a biological system requires recognising the highest number of proteins that are part of this system. These interactions have been examined widely in terms of proteomics recently. It is a branch of science that allows for large-scale analysis, identification, assessment of activity and localisation across multiple biological samples (e.g. cell, tissue, biological flu-ids; Chevalier, 2010). To date a large number of novel pro-teins have been identified including those that are character-istic of a specific physiological and pathological state of an animal. These proteins may help to gain insight into a novel mechanism underlying growth and development of animals (Jayasri et al., 2014; Wright et al., 2014). Proteomic anal-yses have been extensively used to identify diagnostic pro-teins that may be useful as markers for, for example, high animal productivity, assessment of the influence of environ-mental factors, and status of health and disease (Almeida et al., 2015; Janjanam et al., 2014; Kinkead et al., 2015).
As urine is a good source of information on renal func-tion, it is therefore commonly used in diagnostic veterinary
and human medicine. This biological fluid can be collected in large quantities in a non-invasive manner. Physiological urine contains only small amounts of proteins. It should be emphasised that increased urinary protein excretion occurs in both human and animal neonates (neonatal proteinuria) due to increased glomerular permeability and decreased tubular reabsorption (Schafer-Somi et al., 2005; Ojala et al., 2006; O˙zgo et al., 2009). It is estimated that approximately 30 % of all proteins excreted in urine are plasma proteins, whereas the remaining 70 % are proteins derived from the kidney and urinary tract (Thongboonkerd and Malasit, 2005). Extensive effort has been made to find diagnostic urinary protein mark-ers of renal function, especially the glomerulus and the re-nal tubules. Moreover, urinary proteomics has been exten-sively used to not only diagnose pathological processes that affect the excretory system but also to diagnose the functions of the other systems and other organs (Hong et al., 2007; Weissinger et al., 2007; Zimmerli et al., 2008).
al. (2012) found kidney-derived urinary protein biomarkers of acute kidney injury in sheep. Palviainen et al. (2012) pos-tulate that these proteins may serve as a good markers for the early diagnosis of acute kidney injury in humans. The study of Lee et al. (2008) revealed a significant difference between urinary proteome of neonatal and adult rats.
The present paper was undertaken to analyse and iden-tify urinary proteins that were significantly altered in urine of calves in response to short-term administration of milk replacer with lactose addition by using 2-D electrophore-sis combined with matrix-aselectrophore-sisted laser desorption/ionisation and time-of-flight (MALDI-TOF) mass spectrometry.
2 Materials and methods
The experiment was carried out on eight Polish Holstein-Friesian var. Black-and-White male calves in the second week of life. The use and handling of animals for this ex-periment was approved by the Local Commission of Ethics for the Care and Use of Laboratory Animals (Resolution No. 3/2010). The body mass of calves averaged 38 kg on the 13th day of life. During the first three days of life, calves were fed with pooled colostrum and then from the 4th day to 13th day with milk replacer (Mlekovit Imupro®, Polmass) con-taining 23 % crude protein, 16 % crude fat, 0.1 % crude fiber, 45 % lactose, 7.5 % crude ash, 1.7 % lysine, 0.42 % methion-ine, 0.9 % calcium and 0.7 % phosphorus, twice daily in the amount of 10 % of body weight per day. On the 13th day (during the evening feeding) and on the 14th day (during the morning feeding) monohydrate lactose (Pharma Cosmetic) in the amount of 1 g kg−1of body weight was added into the milk replacer.
Blood plasma concentration of potassium was analysed using inductively coupled plasma optical emission spectrom-etry (ICP-OES) with a 2000 DV spectrometer (Perkin Elmer Inc) – the results are presented in the study of Dratwa-Chał upnik et al. (2016b). The blood glucose level was deter-mined spectrophotometrically (Power-Wave Xa, Bio Tek) – the results were previously published in the study of Dratwa-Chałupnik et al. (2016a).
Urine samples were collected directly into the collection bags attached to the loins on the 13th day of life (before lac-tose administration) and on the 14th day of life (after second dose of lactose), 3 h before the evening feeding. The samples were centrifuged (15 min, at 4◦C, 3000 g) and then concen-trated with 3 kDa molecular weight cut-off filters AmiconUl-tra (Millipore) and stored at−80◦
C.
2.1 Two-dimensional electrophoresis (2-DE)
Before 2-DE analysis, urine samples were desalted and cleaned using ReadyPrep™2-D Cleanup Kit (Bio-Rad). Two technical replicates were run for each urine sample.
Processed urine samples were dissolved in the lysis buffer containing 7 M urea, 2 M thiourea, 4 %w/vCHAPS, 1 %w/v
dithiothreitol (DTT), 0.2 %w/v3–10 ampholytes and 2 mM tributyl phosphate (TBP). Urine protein samples (100 µg) were applied to pH 4–7, 7 cm NL ReadyStrip™ IPG strips (Bio-Rad). Urinary proteins were focused according to the following procedure: (i) 250 V for 125 Vh, (ii) 500 V for 250 Vh, (iii) 1000 V for 500 Vh, (iv) linear increase to 5000 V for 1.5 h, (v) 5000 V for 25 000 Vh using a Protean®IEF Cell (Bio-Rad). After IEF, the IPG strips were reduced with DTT in equilibration buffer (6 M urea, 0.5 M Tris/HCl, pH 6.8, 2 %w/v sodium dodecyl sulfate (SDS), 30 %w/v glycerol and 1 %w/vDTT) for 15 min and then alkylated with iodoac-etamide (2.5 %w/v) for 20 min at ambient temperature. The second dimension was performed on 12 % SDS polyacry-lamide gels at 40 V for 1.5 h and then at 100 V for 13 h at 10◦C using a Protean Plus™ Dodeca Cell™electrophoretic chamber (Bio-Rad). After 2-DE separation, the gels were stained with CBB G-250 according to modified Kang method (Pink et al., 2010).
2.2 Image analysis
The gels were scanned using a GS-800™calibrated densito-meter (Bio-Rad). Analysis of 2-D images was performed us-ing a PDQuest Advanced gel analysis software version 8.0.1 (Bio-Rad). Image filtration, spot detection, intensity quantifi-cation, background substraction and spot matching between gels were performed automatically, followed by manual ver-ification to remove false spots and to add missed spots. The spots present on at least three gels before and after adminis-tration of lactose were designated as expressed protein spots and were further analysed. The spot volume (expressed as a spot optical density – OD) was used as the parameter for quantifying protein abundance after normalisation based on the local regression model (LOESS). After normalisation, the volume of each spot was averaged for two replicates of each biological sample. The replicate gels used for match-set making had, at least, a correlation coefficient value of 0.8. To measure the variability before and after administration of lactose, the coefficients of variation were calculated, and ad-ditionally for each protein spot the mean spot quantity value was calculated.
2.3 Mass spectrometry
The protein spots showing statistically significant differences were manually excised from the gels and decolourised by washing in buffer containing 25 mM NH4HCO3in 5 %v/v
Figure 1.2-DE map of the differentially abundant calves urine pro-teins. Presented 2-D gel was stained with Coomassie Brilliant Blue G-250; 100 µg of proteins was applied on the IPG strip (7 cm, pH 4– 7) for the first dimension, and the second dimension was performed on 12 % SDS-PAGE gels. Spot numbers correspond to those in Ta-ble 1.
loaded onto a MALDI-MSP AnchorChip™ 600/96 plate (Bruker Daltonics, Germany). Peptide Mass Standard II was used (mass range 700–3200 Da, Bruker Daltonics) for cal-ibration of the mass scale. Mass spectra were acquired in positive-ion reflector mode using a Microflex™ MALDI-TOF mass spectrometer (Bruker Daltonics, Germany). Pep-tide mass fingerprinting (PMF) data were compared to mammalian databases (SWISS-PROT; http://us.expasy.org/ uniprot/ and NCBI; http://www.ncbi.nlm.nih.gov/) with the aid of MASCOT search engine (http://www.matrixscience. com/). Search criteria included trypsin as an enzyme, car-bamidomethylation of cysteine as a fixed modification, me-thionine oxidation as a variable modification, mass tolerance to 150 ppm and a maximum of one missed cleavage site. The results were further validated by the MASCOT score (only statistically significant hits were applied) and sequence cov-erage.
2.4 Statistics
Significance of the differences in protein abundance pattern was confirmed during image analysis by one-sided Student’s
t test using PDQuest Advanced gel analysis software.
3 Results
Approximately 209 protein spots were detected in each anal-ysed gel, but only spots common for both experimental groups (132) were further analysed. Of these, four showed
significantly decreased abundance: alpha-1-antiproteinase (A1AT), serotransferrin (TF), sex hormone-binding glob-ulin (SHBG) and cytochrome P450 2E1 (CYP2E1). One showed an increased abundance: adenosine triphosphate (ATP)-citrate synthase (Table 1).
An example of a gel demonstrating the urine protein spots that differed in abundance is presented in Fig. 1.
In all calves, loose stools were observed with distinctive dark green colour after the second dose of lactose.
4 Discussion
Following lactose administration five urinary proteins were found differentially expressed. These proteins included alpha-1-antiproteinase, serotransferrin, sex hormone-binding globulin, ATP-citrate synthase and cytochrome P450 2E1.
The decreased abundance of alpha-1-antiproteinase in urine of calves analysed in response to administration of lac-tose was accompanied by increased abundance of this pro-tein in the plasma (Dratwa-Chałupnik et al., 2016a). Alpha-1-antiproteinase belongs to the acute-phase proteins; its level in the blood plasma increases in response to inflammation or infection (Janciauskiene et al., 2011). Presumably due to the involvement of alpha-1-antiproteinase in response to di-arrhea (observed in calves in response to the oversupply of lactose), its removal with the urine was decreased.
T ab le 1. The list of the dif ferentially ab undant calv es urine proteins and characterisation of the parameter identificati on using MALDI-T OF mass spectrometer . Mean relati v e ab undance MALDI-T OF MS Spot Protein name Accession Calculated T heoretical Before SD After SD F old Significance of Mascot Sequence Mass v alues no. no. Mr (kDa) pI/Mr (kDa) lactose lactose change dif ferences score co v erage (%) matched addition addition 1. Cytochrome P450 2E1 O18963 80.10 8.10/56.85 602.02 409.04 134.12 158.58 − 4 . 49 p ≤ 0 . 05 72 21 10 2. Serotransferrin Q29443 80.00 6.75/79.87 1574.36 1081.67 161.30 158.62 − 9 . 76 p ≤ 0 . 05 72 30 17 3. Alpha-1-antiproteinase precursor NP_776307 60.60 6.05/46.42 1846.90 325.65 826.40 772.95 − 2 . 23 p ≤ 0 . 05 98 32 15 4. Se x hormone-binding glob ulin isoform X5 XP_005220374 53.30 5.56/37.66 1919.20 269.57 1383.84 437.28 − 1 . 39 p ≤ 0 . 05 129 54 12 5. A TP-citrate synthase isoform 2 NP_942127 31.05 6.95/120.61 18.90 11.78 935.00 586.27 49.47 p ≤ 0 . 05 79 12 13 Spot number of the identified proteins correspond to those presented on the 2-DE map of calv es urine proteins (Fig. 1).
Sex hormone-binding globulin (SHBG) is another urine protein whose abundance was reduced in response to admin-istration of lactose. This protein transports sex steroids and regulates their access to target tissues. Selva et al. (2007) demonstrated that glucose, sucrose and fructose reduced SHBG production by hepatocytes. The results of this study can indicate that an increased supply of lactose disaccharide may also reduce the synthesis of SHBG; consequently, re-moval of this protein in the urine is reduced. Only a slight increase in plasma glucose level was observed in calves 12 h after the second dose of lactose administration (Dratwa-Chałupnik et al., 2016a).
A significant increase in the abundance of ATP-citrate syn-thase in urine was shown in the calves examined after admin-istration of lactose. Increased removal of ATP-citrate syn-thase in the urine indicates that the filtered protein is not reabsorbed. The main reabsorption site of ATP-citrate syn-thase is proximal tubule, where it is carried out by the apical membrane Na/citrate cotransporter (Levi et al., 1991). Hy-pokalemia in rats, induced by a potassium-poor diet, stimu-lates the Na/citrate cotransporter; consequently, a reduction in urinary citrate excretion is observed (Levi et al., 1991). Melnick et al. (2000) did not observe any changes in the ATP-citrate synthase removal in urine in the infant rats, when potassium-poor diet was applied, despite the decrease in the concentration of this element in the plasma of these animals. A significant increase was observed in the concentration of potassium in the blood plasma of the experimental calves af-ter administration of lactose to milk replacer formula (from 3.89 to 4.08 mmol L−1) – the results are presented in the study of Dratwa-Chałupnik et al. (2016b). Perhaps the in-creased K concentration in the blood plasma of calves exam-ined contributed to increased urinary ATP-citrate synthase excretion. This is consistent with findings in our previous study conducted on older calves reporting no effect of lactose administration on the plasma potassium concentration or on the abundance of ATP-citrate synthase (Dratwa-Chałupnik et al., 2016b).
CYP2E1 in the urine of calves. The reason why glucose in-hibits an abundance of cytochrome P450 2E1 is not clear.
It may be hypothesised that the lactose oversupply was the main factor that influenced the abundance level of the two proteins (sex hormone-binding globulin and cytochrome P450 2E1), and the diarrhea may be the reason responsi-ble for the observed alterations in the abundance of the re-maining three proteins (alpha-1-antiproteinase, serotransfer-rin and ATP-citrate synthase). The results of the present and previous studies clearly indicate that excessive amount of lactose in a diet of calves induces changes not only in blood plasma proteins (Dratwa-Chałupnik et al., 2016a) but also in urinary proteins.
Establishment of the two-dimensional maps reflecting characteristic patterns of urinary proteins of domestic ani-mals might be useful for designing further proteomic stud-ies aimed at elucidating the patterns of renal adjustments to various pathophysiological factors including environmental stressors. Results of such studies may allow the testing of urine samples for proteins that are produced while having a particular disease.
5 Data availability
The original data are available upon request to the corre-sponding author.
Acknowledgements. This work was supported by scientific grant from National Centre of Science, Poland, 2011–2013 (project no. N N311 016239).
Edited by: M. Mielenz
Reviewed by: B. Kuhla and one anonymous referee
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